N-Acetylneuraminic acid (sialic acid) is a 9-carbon acidic monosaccharide serving as terminal residue in a variety of glyco-conjugates, e.g., oligosaccharides, glycoproteins and gangliosides. During the course of lysosomal catabolism of these glyco-conjugates, sialic acid is liberated through the lysosomal membrane to the cytoplasm, where it undergoes further metabolic reactions. In recent years a number of inherited human disorders involving sialic acid has been described which are characterized by the excessive intralysosomal accumulation of free sialic acid. To investigate the possibility that this accumulation is due to defective passage of the monosaccharide across the lysosomal membrane, we have measured the rate of loss of free sialic acid from lysosome-rich granular fractions prepared from cultured fibroblasts of affected patients as well as from normal individuals. Loading of normal fibroblast granular fractions with free sialic acid was achieved by prior incubation of intact cells for 2-4 days with high concentrations (20-75 mM) of N-acetyl-D-mannosamine (ManNAc), a metabolic precursor of sialic acid. No loss of free endogenous or ManNAc-derived sialic acid could be detected from granular fractions of mutant cells, whereas rapid loss (t-1/2 12 min) was observed fromtheloaded normal granular fractions. These observations support the notion that free sialic acid storage disease is the result of an impaired mechanism of passage of sialic acid across the lysosomal membrane and may provide a second example, human cystinosis being the first, of a lysomal storage disorder due to defective lysosomal transport.